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Determination of Smoltification Status in Juvenile

Migratory Rainbow Trout and Chinook Salmon inMinnesotaMary T. Negus

a

a Minnesota Department of Natural Resources, Division of Fisheries, 5351 North Shore Drive,

Duluth, Minnesota, 55804, USA

Version of record first published: 08 Jan 2011.

To cite this article: Mary T. Negus (2003): Determination of Smoltification Status in Juvenile Migratory Rainbow Trout and

Chinook Salmon in Minnesota, North American Journal of Fisheries Management, 23:3, 913-927

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North American Journal of Fisheries Management 23:913–927, 2003 Copyright by the American Fisheries Society 2003

Determination of Smoltification Status in Juvenile Migratory

Rainbow Trout and Chinook Salmon in Minnesota

MARY T. NEGUS* Minnesota Department of Natural Resources, Division of Fisheries,

5351 North Shore Drive, Duluth, Minnesota 55804, USA

Abstract.— The progress of smoltification was quantified with an enzyme assay (gill sodium-

potassium-activated adenosine triphosphatase, or ‘‘Na,K-ATPase,’’ activity) in nonnative pop-

ulations of migratory salmonines that are stocked or naturally spawned in Minnesota tributaries

of Lake Superior. This information was needed to develop stocking strategies that would maximize

imprinting at intended locations and to determine the causes and consequences of early emigration

in stream-reared fish. Species and strains tested included (1) chinook salmon Oncorhynchus tshaw-

ytscha during hatchery rearing and after stocking, (2) two strains of O. mykiss—steelhead (natu-

ralized) and a Kamloops (hatchery) strain of migratory rainbow trout—during hatchery rearing

and after stocking, and (3) stream-reared steelhead emigrants captured in smolt traps. The ATPase

activity level that distinguished smolts from nonsmolts was 11 mol Pi · (mg protein)1 · h1 for

chinook salmon and 10 mol Pi · (mg protein)1 · h1 for all groups of rainbow trout tested. Plots

of ATPase activity against fork length, weight, and body depth indicated that 91% of chinook

salmon and 100% of rainbow trout were stocked at sizes that exceeded a minimum size for smolting.

Temperature appeared to influence smoltification of chinook salmon. Stocking into a river signif-

icantly increased ATPase activity levels of hatchery chinook salmon and Kamloops rainbow trout,

but data were inconclusive for steelhead. Most stocked steelhead fry and naturally spawned steel-

head that emigrated before age 2 were not smolts and may have emigrated as a result of natural

exploratory movement. My results suggest that stocking all fish in May or June, controlling rearing

and stocking temperatures of chinook salmon, and controlling the sizes attained by migratory

rainbow trout in the hatchery may maximize in-stream smolting and future homing to desired

locations.

Chinook salmon Oncorhynchus tshawytscha

(naturalized) and two strains of O. mykiss—natu-

ralized steelhead and hatchery-reproduced Kam-

loops rainbow trout—are anadromous fish native

to the Pacific coast that have been stocked into

Minnesota tributaries of Lake Superior (Close et

al. 1984; Krueger et al. 1994; Peck et al. 1994).

Broodstock of fall-spawning chinook salmon

(Washington State origin, smolting at age 0) and

spring-spawning rainbow trout of both strains are

captured annually in French River for propagation

at the French River State Fish Hatchery. Age-0chinook salmon, yearling steelhead, and yearling

Kamloops rainbow trout (hereafter Kamloops) are

stocked into Minnesota tributary streams in spring

at ‘‘smolt size,’’ a loosely applied term based on

size and color change in some fish. These fish live

entirely in freshwater and are therefore called mi-

gratory rather than anadromous, but their original

life history patterns, including smoltification, per-

sist. Smoltification is the primary time for olfac-

tory imprinting, although fish may also remember

* E-mail: [email protected]

Received September 25, 2001; accepted January 8, 2003

odors experienced before that stage (Hasler and

Scholz 1983; Morin and Døving 1992; Quinn

1993; Pascual et al. 1995; McCormick et al. 1998).

To effectively enhance stocks that will home to

specific locations where angling opportunities are

desired, hatchery fish must be stocked before im-

printing is completed.

Increased salinity tolerance is another charac-

teristic of smoltification, and most studies of smol-

tification have involved populations that migrate

to saline environments (Wedemeyer et al. 1980;

Folmar and Dickhoff 1981). Although salinity tol-erance is obviously irrelevant for smolts in Lake

Superior, the physiological changes still occur and

provide a means of pinpointing smoltification (and

indirectly, the period of imprinting). Gill sodium-

potassium-activated adenosine triphosphatase

(Na,K-ATPase) activity (hereafter called ‘‘ATPase

activity’’), which is strongly correlated with salin-

ity tolerance, can be used to quantify progress of

smoltification (Zaugg 1982; Boeuf and Prunet

1985; McCormick et al. 1987; Johnson et al. 1991;

Schrock et al. 1994). Superficial characteristicscommonly recognized as indicators of smoltifi-

cation (e.g., coloration, body size, condition factor,

and behavior) are imprecise measures of the peak

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914 NEGUS

of this life stage and may vary with species, pop-

ulation, or locale (Zaugg and McLain 1972; Vir-

tanen et al. 1991; Beeman et al. 1995; Haner et al.

1995).

Rainbow trout and chinook salmon stocks inMinnesota waters of Lake Superior have been de-

clining in recent years, and factors contributing to

this decline are under investigation (Schreiner

1995). Despite potential negative effects of pro-

longed hatchery rearing on imprinting and smol-

tification (Wagner et al. 1963; Wedemeyer et al.

1980; Quinn 1993), there is public pressure in

Minnesota to rear all hatchery stocks to the largest

possible sizes before stocking. Adult chinook

salmon, steelhead, and Kamloops routinely stray

to other rivers along the Minnesota shoreline and

beyond, suggesting that they partially imprint inthe hatchery or fail to imprint to their stocking

sites (Jones and Schreiner 1997; Peck et al. 1999;

D. R. Schreiner, Minnesota Department of Natural

Resources, personal communication). Even when

steelhead are stocked as fry (as in French River)

or are naturally produced (as in Knife River), the

young fish often display counterproductive behav-

ior by emigrating at small sizes (before age 2) that

experience low survival (Olsen et al. 2000; Spur-

rier et al. 2000a). Determining the smolt status of

these small emigrants may help to explain thecause of early emigration and the reasons for their

high mortality.

The objectives of this study were to (1) evaluate

smoltification readiness in hatchery chinook salm-

on, steelhead, and Kamloops, (2) determine wheth-

er smoltification is stimulated when fish are

stocked into a stream, (3) identify the smoltifica-

tion status of emigrating steelhead (naturally

spawned or stocked as fry) captured in smolt traps,

and (4) determine whether ATPase activity was

related to size, coloration, condition, water tem-

perature, and date of capture to identify indicators

of smoltification applicable to local stocks.

Methods

Study area and fish populations.—I tested chi-

nook salmon, steelhead, and the Kamloops strain

of rainbow trout being reared at the French River

State Fish Hatchery. The hatchery outflow water

enters the French River (Figure 1) about 100 m

upstream from the mouth. The hatchery-reared

stocks were destined for various Lake Superior

tributaries, where they were expected to imprintand supplement fishing opportunities during

spawning runs (Figure 1). I also tested stream-

reared steelhead that had been stocked as fry in

the French River or naturally spawned (wild) in

the Knife River and that were later captured in

smolt traps located in these rivers.

Minnesota’s shoreline of Lake Superior contai ns

54 tributary rivers or streams with habitat for mi-gratory species (MNDNR 1992). These tributaries

are generally much smaller and less productive

than West Coast rivers where chinook salmon and

rainbow trout originated. The Knife River system

is the only tributary that is accessible to migratory

fish for its entire 113-km length. The remaining

53 streams total about 62 km of habitat, but 25 of

these streams have marginal habitat due to their

small size. The steep landscape in Minnesota’s

North Shore region of Lake Superior forms a bar-

rier to upstream migration of fish in most streams,

and waterfalls often occur only a few meters up-stream from the lake. In general, the streams have

little groundwater input and are subject to high

spring runoff and widely fluctuating flows. Flows

in French and Knife rivers, for example, often

range from 0.1 to 1 m3 /s in summer but can spike

to 6–28 m3 /s or more duri ng storms. Cold winter

temperatures can cause anchor ice, ice dams, and

dry streambeds in some locations in some years.

Warm, dry weather can severely reduce flows and

heat the water, and heavy rainfall or snowmelt can

rapidly increase flows to flood stage. Thesestreams have low productivity and a limited car-

rying capacity for fish. Brook trout Salvelinus fon-

tinalis inhabit the headwaters of most streams, and

lower reaches contain a few brown trout Salmo

trutta, sculpins Cottus spp. and minnows. These

lower reaches are stocked with steelhead fry,

whereas age-0 chinook salmon and yearling rain-

bow trout are generally stocked a short distance

above or below the barriers. Since 1992, Kamloops

stocking has been restricted to three tributaries

near the city of Duluth (MNDNR 1992; Figure 1)

to reduce the probability of interbreeding with nat-

uralized steelhead.

Fish sampling.—To determine whether smolti-

fication was occurring in hatchery stocks (objec-

tive 1), ATPase activity was measured in chinook

salmon, steelhead, and Kamloops during hatchery

rearing to smolt size (Table 1). Hatchery-reared

chinook salmon achieved smolt size at age 0

(reared from the January hatch to May or June),

and the earliest samples were taken in March.

Steelhead were sampled periodically from Novem-

ber at age 0 to May at age 1; Kamloops weresampled from November at age 0 through Septem-

ber at age 1. Ordinarily, 36 samples (1 sample/

fish) were taken from each species or strain on

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915DETERMINATION OF SMOLTIFICATION STATUS

FIGURE 1.—Minnesota’s Lake Superior shoreline, known as the North Shore, including tributaries into which

migratory species are stocked. Letters in circles preceding river names indicate species or strains stocked into those

rivers: C chinook salmon, S steelhead, and K Kamloops strain of rainbow trout. All migratory fish stocked

into Minnesota waters are reared at the French River State Fish Hatchery.

each sampling date. Hatchery year-classes were

1997, 1998, and 1999 for chinook salmon; 1996

and 1997 for steelhead; and 1996, 1997, and 1998

for Kamloops. The 1997 year-class of chinook

salmon was reared with two different thermal re-

gimes: normal rearing temperatures of 5–7C and

accelerated growth temperatures of 10–12C. Chi-

nook salmon rearing temperatures in 1998 were

also elevated (9–12C) to speed growth, and rear-

ing temperatures in 1999 resembled the normal

temperatures of 1997. The ATPase activities of all

fish were measured to determine whether smolting

(indicated by an elevation in ATPase activity

above threshold values) was occurring duringhatchery residency.

To determine whether stocking into flowing wa-

ter stimulated smolting (objective 2), samples were

taken from hatchery-reared chinook salmon, steel-

head, and Kamloops before and after stocking (Ta-

ble 1). No individuals were sampled more than

once. A random sample of hatchery fish was

stocked about 1 km above the French River smolt

trap so they could be recaptured, and fish remain-

ing in the hatchery were sampled on the same day

to represent prestocked fish. Mean ATPase activ-

ities before and after stocking were compared us-

ing pooled-variance t -tests ( 0.05).

To determine the smoltification status of stream-

reared steelhead emigrants (objective 3), steelhead

that had been stocked as fry in French River or

naturally reproduced in Knife River were sampledfrom smolt traps in these rivers. Samples were

taken when at least 3–5 fish/d were being trapped.

Up to 36 fish were sampled per day; fish less than

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916 NEGUS

TABLE 1.—Salmonid populations sampled for gill sodium-

potassium-activated adenosine triphosphate activity. There

was one sample per fish and one assay per sample.

Species or strainNumber

of samples

Hatchery-reared fish

(sampled periodically up to day of stocking)

Chinook salmon

Steelhead

Kamloops

242

343

459

Hatchery fish stocked into French River

(sampled at recapture)

Chinook salmon

Steelhead

Kamloops

98

38

36

Stream-reared fish

(sampled when captured in the river’s trap)

Fry-stocked steelhead (French River)Wild steelhead (Knife River)

368122

80 mm fork length (FL), which were difficult to

sample nonlethally and were always parr, were

omitted when large numbers were captured. The

ATPase activities of these two groups of stream-

reared fish were measured to determine the pres-

ence of smolting.

To determine whether ATPase activity in local

stocks could be related to a more obvious, easilymeasured characteristic (objective 4), ATPase ac-

tivities were plotted against FL (a more consistent

measure than total length because hatchery fish

had obvious fin erosion), weight, body depth, con-

dition factor (105 · weight/fork length3), and sam-

pling date; ATPase activities were also compared

with body color (distinguished by different sym-

bols). The threshold level in ATPase activity re-

quired for smoltification was defined as the upper

end of the range of ATPase activities detected in

the smallest fish tested that had dark bands and

swam strongly against the current (chinook salmon

sampled in March and April and rainbow trout

sampled in November and January). Recommend-

ed guidelines were 0 mol to about 10–12 mol

Pi · (mg protein)1 · h1 for nonsmolting juveniles

and 12–15 mol Pi · (mg protein)1 · h1 or more

for smolting juveniles (R. Schrock, USGS, West-

ern Fisheries Research Center, personal commu-

nication), although these levels vary by species,

procedure, and laboratory. Ewing et al. (1980) dis-

cussed the concept of size thresholds for smolti-

fication; in my study, I quantified minimum sizesfor smoltification in chinook salmon, steelhead,

and Kamloops by visually inspecting the plots of

ATPase activity as a function of fork length and

then by estimating the minimum size at which el-

evated ATPase activities (indicating the onset of

smoltification) were first evident. The ATPase ac-

tivities of three color categories for each species

or strain were analyzed using analysis of variance(ANOVA; 0.05) and posthoc Bonferroni

pairwise comparisons. Water temperatures, FL,

and ATPase activities were plotted against date to

reveal the temporal sequence of changes in all spe-

cies and strains.

ATPase activity assay methods.—At sampling,

each fish was anesthetized and a portion of gill

filament was excised using techniques described

by McCormick (1993) and Schrock et al. (1994).

Each tissue sample (one sample per fish) was

placed in SEI (sucrose, disodium EDTA, imidaz-

ole) buffer solution, numbered, and frozen in liq-uid nitrogen within 30 min. The samples were later

transferred to a 80C freezer until analysis. Each

fish was weighed (g); measured (mm) for fork

length, total length, and body depth; and catego-

rized by body coloration (banded, intermediate, or

silver). The ‘‘banded’’ designation meant that all

parr marks were visible. The ‘‘intermediate’’ des-

ignation meant that parr marks closest to the head

were gone, but marks were still visible near the

caudal fin. The ‘‘silver’’ designation meant that all

parr marks were gone, or those remaining near thecaudal fin were extremely pale. Categorization was

done indoors or out of direct sunlight to reduce

reflection that could affect perceived body color.

Water temperatures in the river and in the hatchery

were continuously monitored. All live fish were

released after sampling.

Gill filament samples were prepared following

the protocol of Schrock et al. (1994), with a few

modifications described here. The samples were

extracted in SEI only, eliminating the solution con-

taining deoxycholate, a detergent additive that was

found to be unnecessary. Samples were ground

with a Tissue Tearor and then centrifuged for 8

min at 2,000 relative centrifugal force (RCF). Su-

pernatant was pipetted off, SEI was added to the

pellet, and each vial was vortexed on the high set-

ting for 10 s. Samples were sonicated for 4–5 con-

tinuous seconds using a Sonics and Materials, Inc.,

Vibra Cell 50-W Ultrasonic Processor set at Out-

put 20 and centrifuged for 10 min at 12,000

RCF; 150–200 L of the enzyme preparation su-

pernatant was transferred to a culture tube. Rep-

licates of each sample, references, and standardswere included in each microassay plate to reduce

the probability of incorrect values due to pipetting

errors. Values of the replicates were averaged.

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TABLE 2.—Sodium-potassium-activated adenosine triphosphate (ATPase) activities that defined the threshold of smolt-

ing for chinook salmon and migratory rainbow trout. Minimum sizes were the approximate lengths, body depths, or

weights at which elevated ATPase activities were first measured.

Species orstrain

Rearinglocation

ATPaseactivitya

Minimum sizes for smolting

Fork length(mm)

Weight(g)

Bodydepth(mm)

Approximatenumber

per pound (kg)b

Chinook salmon

Steelhead

Kamloops rainbow trout

Steelhead (fry-stocked)

Wild steelhead

Hatchery

Hatchery

Hatchery

French River

Knife River

11

10

10

10

10

71

150

150

140

140

4

40

40

28

28

15

30

30

25

25

100 (220)

10 (22)

10 (22)

a That is, mol Pi (mg protein)1·h1; the first whole integer above the range of values measured in the smallest groups of juveniles

tested.b Number per pound is a common hatchery measurement. Because these fish cannot be blotted effectively when held in a net and because

the minimum weight is a conservative estimate for the low end of smolting, the number of fish has been reduced slightly to provide a

rough guideline for hatchery managers.

The evaluation procedure consisted of two as-

says: one to measure the amount of inorganic phos-

phate (Pi) liberated by the action of ATPase and

one to measure the amount of protein. The phos-

phate assay followed the procedure of Schrock et

al. (1994). The Bio-Rad protein assay was substi-

tuted for the more complicated protein assay used

by Schrock et al. (1994). All phosphate and protein

samples were read at 590 nm on a Biolog Mi-

crostation 96-well plate reader. The ATPase activ-

ity was reported as mol Pi

· (mg protein)1 · h1.

Results

Smoltification Readiness in Hatchery Fish

Elevated ATPase activity levels indicative of

smolting were found in many hatchery fish, but

smoltification was obviously not an all-or-nothing

event; nonsmolts were sampled at nearly every

size and date that smolts were sampled. The thresh-

old ATPase activity for smolting in chinook salm-

on was determined to be 11 mol Pi · (mg pro-

tein)1 · h1, because 10.6 was the highest activity

found in the smallest chinook salmon sampled (Ta-

ble 2; Figures 2, 3). The threshold ATPase activity

for smolting in rainbow trout was determined to

be 10 mol Pi · (mg protein)1 · h1 because 9.8

was the highest activity found in the smallest rain-

bow trout sampled (Table 2; Figures 4, 5, 6).

Stimulation of smoltification between stocking

and recapture

Mean ATPase activity between the time of

stocking and recapture increased significantly in

chinook salmon (t 3.704, df 146, P 0.001)and Kamloops rainbow trout (t 3.003, df

101, P 0.003) but decreased significantly in

steelhead (t 4.396, df 66, P 0.001; Table

3). Only one stocking trial was run with steelhead,

and that group was the only one with a mean

ATPase activity level that exceeded the threshold

for smoltification before stocking (Table 3). Most

stocked chinook salmon emigrated immediately,

and the highest and lowest ATPase activities were

measured in those fish recaptured first. Most of the

later recaptures remained in the stream until smolt-

ing (Negus 2000). The largest numbers of chinook

salmon with low ATPase activities were recaptured

1 d after stocking at low temperatures (below 9 C).

Emigration by stocked steelhead and Kamloops

was more gradual, and about 50% left within 1

week of stocking.

Smoltificationt Status of Fry-Stocked and Wild

Steelhead Emigrants

Elevated ATPase levels indicative of smolting

were measured in some steelhead emigrants cap-

tured on every sampling date in spring, but none

were found in fall samples (Figures 7, 8). Tem-

peratures during all spring and fall periods ex-

ceeded normal hatchery rearing temperatures forsteelhead and Kamloops. Most smolting emigrants

were age-2, or very large age-1 fish. Only one

emigrant in the French River exceeded 244 mm

FL, and the largest emigrant in Knife River was

214 mm FL.

Relationship between ATPase Activity and Other

Measurements

The smallest sizes at which smoltification was

detected were ascertained for each of the strains.

The minimum size of smoltification in chinook salmon (ATPase activity 11 mol Pi · [mg pro-

tein]1 · h1) was 71 mm FL, as seen in plots of

all year-classes combined (Figure 3; Table 2). A

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918 NEGUS

FIGURE 2.—Temporal changes in daily water temperature, sodium-potassium-activated adenosine triphosphatase

(ATPase) activity, and fork length of four groups (three year-classes) of chinook salmon sampled during rearing

in Minnesota’s French River State Hatchery. Error bars have inner tick marks denoting means SEs and outer

‘‘T’’ marks denoting means SDs. In 1997, temperatures were not recorded after early June because most chinook

salmon were stocked by that time. The last groups sampled that spring had been held for 2 weeks at approximately

10–12C. Triangles identify dates when fish were sampled just before stocking trials.

second, even greater increase in ATPase activities

occurred above 15 mol Pi · (mg protein)1 · h1 in

the largest fish from the 1997 accelerated growth

group that was reared at higher temperatures (Fig-

ure 3). The minimum size at which smoltification

occurred for rainbow trout varied depending on

rearing location. Kamloops and steelhead in the

hatchery exhibited elevated ATPase activities at

about 150 mm FL (Figure 6; Table 2). Stream-

reared steelhead appeared to begin smolting at

smaller sizes than fish in the hatchery, roughly

around 140 mm FL. At the time of stocking, 91%

of the chinook salmon and 100% of steelhead and

Kamloops exceeded minimum smolting sizes (Fig-

ures 2, 4, 5). The ATPase activity was not signif-

icantly related to condition factor of any hatcheryor stream-reared stocks tested (Negus 2000).

Elevated ATPase levels were rare in hatchery

Kamloops larger than 260 mm FL, 200 g, and 60

mm body depth (Figure 6; Negus 2000), but these

large nonsmolts were found primarily in summer,

after the normal smolting season. Less than 1% of

the hatchery steelhead exceeded these large sizes

at the time of stocking, but between 21% and 36%

of the Kamloops exceeded at least one of these

measurements when stocked.

Color determination of fish was somewhat sub-

jective, and visi bility of parr marks depended upon

light intensity and viewing angle. Chinook salmon

were especially difficult to classify, so color cat-

egorizations of this species were dismissed. The

ATPase activities differed significantly (P 0.05)

between color categories of hatchery steelhead

(F 2,377 80.940), hatchery Kamloops (F 2,491

21.824), fry-stocked steelhead in French River(F 2,363 68.642), and naturally reproduced steel-

head in Knife River (F 2,118 50.977; Table 4),

although posthoc Bonferroni pairwise compari-

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FIGURE 3.—Individual ATPase activity levels versus

fork length of all three year-classes of age-0 chinook

salmon measured during their rearing periods in Min-

nesota’s French River State Hatchery. The vertical line

delineates the approximate minimum size at which el-

evated ATPase activities were measured in some fish.

sons demonstrated no difference between the in-

termediate and silver color categories of Kam-

loops. In all other cases, dark-banded rainbow

trout had the lowest and silver-colored rainbow

trout had the highest ATPase activities.

Evidence of smolting was seen primarily in chi-

nook salmon reared at or above 9 C and occurred

as early as April (Figures 2, 3). Elevated ATPase

activities were seen in hatchery steelhead and

Kamloops by late February or early March (Fig-

ures 4, 5). The ATPase activities of Kamloops in

the hatchery were depressed after the temperature

reached 12C, but these fish were the largest rain-

bow trout tested (late summer; Figure 5). Stream-

reared steelhead could not be sampled before April

because of ice cover; most fish with elevatedATPase levels emigrated in May or June, and

ATPase activities in French River were depressed

from June and through the fall (Figures 7, 8).

Discussion

Smoltification Readiness in Hatchery Fish

Steelhead, Kamloops rainbow trout, and most

chinook salmon are currently held in the French

River State Fish Hatchery beyond the size when

smoltification can begin. Some Kamloops may be

held past the period of smoltification and regres-sion. Holding fish beyond the start of smoltifica-

tion is not necessarily harmful to the fish, but

hatchery imprinting or sequential imprinting may

explain some of the straying back to French River

by stocked chinook salmon, steelhead, and Kam-

loops. This straying reduces fishing opportunities

at the stocked streams and causes an inflated es-

timate of survival based on counts taken at FrenchRiver. Pascual et al. (1995) suggested that down-

stream emigration during the appropriate season

and physiological state is important to smolt trans-

formation and imprinting, but fish may also re-

member odors experienced before this time. Ma-

turing salmon tend to reverse the sequence of their

outward emigration as juveniles, returning first to

the odors of their release site and continuing on

to the rearing site (hatchery) if its odors can be

detected (Quinn et al. 1989). If there is no stimulus

for further migration once they reach the release

site, the fish may remain there (Dittman and Quinn1996). Seelbach et al. (1994) found that although

smaller steelhead smolts and those stocked farther

upstream in a Lake Michigan tributary suffered

higher mortality than larger smolts and those

stocked into river mouth, those stocked into the

river mouth strayed more. He surmised that min-

imizing straying by stocking upstream outweighs

the costs of higher mortality, when the goal is to

create fisheries in specific locales. The issue of

straying is also important where there is a desire

to maintain spatial segregation between natural-ized and hatchery stocks. Hatchery Kamloops will

spawn with steelhead in a stream setting, but off-

spring having one or more Kamloops parents have

reduced fitness (Close 1999; Negus 1999). Al-

though stocking of Kamloops has been restricted

to reduce the spatial overlap with the naturalized

steelhead during spawning runs, this strategy may

be less effective than desired.

Stimulation of Smoltification between Stocking

and Recapture

Some smoltification was stimulated by place-

ment of chinook salmon and Kamloops in a flow-

ing stream. Although the stocked steelhead did not

respond in a similar way, data were insufficient to

prove that steelhead cannot also be stimulated to

smolt by stocking. Most (93%) of the chinook

salmon were recaptured immediately after stock-

ing. These immediate recaptures included both

smolts and presmolts, whereas those recaptured

later were primarily smolts. The initial mass em-

igration, possibly a schooling behavior, was es-

pecially noticeable after the May 1997 stockingwhen water temperature was about 7C, a tem-

perature that apparently discouraged chinook

salmon smoltification in this study. About 50% of

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FIGURE 4.—Temporal changes in daily water temperature, ATPase activity, and fork length of two year-classes

of steelhead sampled during rearing in Minnesota’s French River State Hatchery. Error bars have inner tick marks

denoting means SEs and outer ‘‘T’’ marks denoting means SDs. The triangle identifies the date when fish

were sampled just before the stocking trial.

the steelhead and Kamloops yearlings that were

stocked and recaptured emigrated within 1 week.

Seelbach and Miller (1993) found that hatchery

steelhead smolts with a mean total length of 195

mm (about 187 mm FL; Negus 2000) stocked in

a Michigan tributary to Lake Superior emigrated

at a similar rate and strayed widely.

Smoltification Status of Stream-Reared Steelhead

Emigrants

Early emigration by steelhead has sometimes

been attributed to early smolting, displacement due

to high water, intraspecific competition, and self-

thinning (Grant 1993; Marschall and Crowder

1995), as well as natural exploratory movement.

Early smolting was not found in most small

stream-reared steelhead in this study, because most

of the emigrants under age 2 were not actuallysmolting. High water was also rejected as an ex-

planation for most premature emigration because

capture in the Knife River trap has been signifi-

cantly correlated with lower flows and captures are

rare during spate flows (Morse and Olsen 1999).

Self-thinning due to intraspecific competition im-

plies that the streams are being stocked or naturally

recruited above their carrying capacity for age-1

parr. Estimating the amount of intraspecific com-

petition is beyond the scope of this study, but data

from the Little Knife River (a branch of Knife

River), show that the number of age-1 emigrants

is directly correlated with the number of age-2

emigrants from the same year-class (Spurrier et al.

2000b; Figure 9). Differing carrying capacities for

ages 1 and 2 would be unlikely to produce such a

direct relationship between the two age-classes.

However, large year-classes or better survival to

age 1 in some years may result in more survivors

and more emigrants throughout the period of

stream residency. Leider et al. (1986) found thatsurvival of steelhead appeared to depend on avail-

ability of suitable rearing environments in down-

river areas because many presmolt steelhead in a

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FIGURE 5.—Temporal changes in daily water temperature, ATPase activity, and fork length of three year-classes

of a Kamloops strain of rainbow trout sampled during rearing in French River State Hatchery. Error bars haveinner tick marks denoting means SEs and outer ‘‘T’’ marks denoting means SDs. Triangles identify dates

when fish were sampled just before stocking trials.

Washington tributary stream moved down to the

main stem, some later moved back upstream, but

many stayed in the main stem until smolting the

following year. Minnesota’s high-gradient tribu-

taries preclude upstream movement by parr, and

these streams are generally much shorter and

smaller than the rivers in their native range. I sug-

gest that natural exploratory movement results in

the premature emigration of parr that quickly reach

the cold, predator-inhabited waters of Lake Su-

perior at a very vulnerable size, and prospects for

survival are poor.

Relationship between ATPase Activity and Other

Measure ments

External features failed to verify smoltification

with certainty, but minimum size, coloration, and

time of year can bracket when smoltification is

imminent. Minimum size represents a size atwhich smoltification may begin under the right

conditions. Minimum sizes similar to those found

in this study (71 mm FL for chinook salmon, 140–

150 mm FL for rainbow trout) have been reported

for age-0 chinook salmon (80 mm FL; Ewing et

al. 1979) and for steelhead (140–160 mm total

length; Chrisp and Bjornn 1978). Once hatchery

fish exceed this size, stocking should be consid-

ered if homing to locations away from the hatchery

is desired, especially if the hatchery outflow enters

a stream that will be accessible to the stocked fish

when they mature. Stream-reared age 2 steelhead

displayed elevated ATPase activity levels at small-

er sizes than hatchery yearling steelhead and Kam-

loops, which may be related to the age differential

or exposure to flowing water and other natural

stimuli in the stream.

Nearly all hatchery Kamloops larger than 260

mm FL, 200 g, and 60 mm body depth and held

into late summer regressed to a presmolt condition,

revealing the transient nature of smoltification in

fish that are retained in a hatchery for some weeksafter the usual time of migration (Fessler and Wag-

ner 1969; Zaugg and McLain 1972; Hoar 1976;

Pascual et al. 1995). Not surprisingly, all but one

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FIGURE 6.—ATPase activity levels versus fork length of individual migratory rainbow trout (two strains reared

in the hatchery and two groups reared in streams), including samples collected in all years but separated by three

body color characteristics (banded, intermediate, and silver). The hatchery-reared fish were tested during the period

of hatchery residency up until the time of stocking; the stream-reared fish were tested at the time of capture in

smolt traps. Vertical lines indicate minimum sizes at which elevated ATPase activities were measured in some fish.

stream-reared fish emigrated at smaller sizes. The

percentage of Kamloops larger than 260 mm FL

dropped from 21% before stocking to 14% at re-

capture in the French River, suggesting differential

survival or a reduced tendency toward emigration.

Partridge (1986) found that steelhead larger than

260 mm FL have a tendency to residualize (remain

in the river).

Color change was not necessarily a precise in-

dicator of smolting for individual fish, a finding

similar to those of Zaugg and McLain (1972) and

Virtanen et al. (1991). Condition factor was not agood predictor of smolting in any of the Minnesota

stocks tested, a finding supported by Beckman et

al. (1999), who stated that changes in condition

factor occur for a variety of reasons unrelated to

smolting. In contrast, condition factor has been

directly correlated with smoltification and emigra-

tion for some steelhead in Washington and Oregon

(Fessler and Wagner 1969; Zaugg and McLain

1972; Beeman et al. 1995; Tipping and Byrne

1996).

Temperatures of 13C and above have been

found to inhibit smoltification in steelhead (Wed-

emeyer et al. 1980), and rearing temperatures of

steelhead and Kamloops at the French River State

Hatchery were kept below 12C at all times, exceptfor Kamloops during summer 1997. In contrast,

stream temperatures frequently exceeded 12C

during the spring emigration period. Elevated

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TABLE 3.—Samples of chinook salmon, Kamloops,

and steelhead taken after stocking and recapture in the

French River smolt trap. Recapture of emigrating fish

stocked in spring 1999 was curtailed when a severe

flood interrupted trap operation on 4 July. Sodium-po-

tassium-activated adenosine triphosphate (ATPase) ac-tivity is reported in mol Pi·(mg protein)

1·h1 .

Numberstocked

Recaptured

Number PercentStocking

date

Mean ATPase activity

Beforestocking

Afterrecapture

Chinook salmon, age 0

100

50

50

50

98

27

16

28

98

54

32

56

14 May 1997

8 Jun 1998

17 May 1999

26 May 1999

6.0

7.4

6.5

9.1

10.0

13.0

13.2

Steelhead yearlings

50 38 76 1 Jun 1998 12.4 8.3

Kamloops rainbow trout yearlings

50

50

41

7

82

14

17 May 1999

28 Jun 1999

9.2

8.0

10.6

10.1

FIGURE 7.—Temporal changes in daily water temperature, ATPase activity, and fork length of steelhead that had

been stocked as fry in the French River, reared in the stream, and captured at the smolt trap as they emigrated

downriver in spring and fall. Error bars have inner tick marks denoting means SEs and outer ‘‘T’’ marks denotingmeans SDs. Each fish was sampled only once at the time of capture in the trap, then replaced in the river below

the trap. Note that a maximum of 36 fish were sampled per day and that we eliminated small fish (80 mm FL)

when larger fish were available.

ATPase activities were not found in hatcher y Kam-

loops after the temperature exceeded 12C, and

ATPase activities of stream-reared steelhead were

depressed in June and in the fall following periods

of elevated temperatures. The elevated tempera-tures used for rearing some chinook salmon in this

study ranged from 10C to 12C, which is reported

to be favorable for growth and survival of this

species (Beckman et al. 1999).

Smoltification in age-0 chinook salmon appears

to be related to spring rearing temperature, al-

though other studies have implicated spring

growth rate as a controlling factor for yearling

chinook salmon in Washington and Oregon (Beck-

man et al. 1998, 1999). Although the Minnesota

chinook salmon reared at warmer temperatures

tended to grow faster, even the largest (fast grow-

ing) fish in cooler water had low ATPase activities,

so spring growth rate had less effect than rearing

temperature. Pascual et al. (1995) found that chi-

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924 NEGUS

FIGURE 8.—Temporal changes in daily water temperature, ATPase activity, and fork length of steelhead that had

been naturally spawned in the Knife River, reared in the stream, and captured at the smolt trap as they emigrateddownriver in spring. Error bars have inner tick marks denoting means SEs and outer ‘‘T’’ marks denoting means

SDs. Each fish was sampled only once at the time of capture in the trap, then replaced in the river below the

trap. Note that a maximum of 36 fish were sampled per day and that we eliminated small fish ( 80 mm FL) when

larger fish were available.

TABLE 4.—Mean ATPase activities ( N ; SE) in different color phases of each strain of migratory rainbow trout tested.

Chinook salmon have not been included here because color differentiation was more ambiguous.

StrainRearinglocation

Color phase

Banded Intermediate Silver

SteelheadKamloops

Steelhead (fry-stocked)

Wild steelhead

HatcheryHatchery

French River

Knife River

5.6 (148; 0.197)6.2 (123; 0.261)

5.8 (239; 0.201)

6.1 (53; 0.343)

9.4 (129; 0.317)8.4 (116; 0.260)

8.2 (89; 0.445)

10.6 (41; 0.687)

10.5 (103; 0.384)8.3 (255; 0.207)

12.9 (38; 0.746)

14.7 (27; 0.847)

nook salmon released into the Columbia River dur-

ing low flows and high temperature produced the

lowest straying rates, possibly because fish stayed

in the rivers longer, imprinting was facilitated at

high temperatures, or the odors for olfactory im-

printing were more concentrated. Results of my

study support the possibility of enhanced chinook

salmon smoltification at 10–12C.

Stream-reared steelhead emigrated primarily

from early May through June, so this may be the

most appropriate time for stocking. Emigration

timing by naturally spawned chinook salmon in

Minnesota waters is unknown (and spawning hab-

itat is limited), but chinook salmon in the Brule

River, a Lake Superior tributary in Wisconsin,

smoltify primarily in May and June of their first

year (DuBois and Pratt 1994).

Management Implications

Hatchery chinook salmon and migratory rain-

bow trout that exceed minimum sizes and have

intermediate or silver coloration in May or June

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FIGURE 9.—Stream-reared (naturally spawned) steel-

head emigrants captured in the Little Knife River smolt

trap. Each point represents the total number of fish from

each year-class (1987–1999) that emigrated at ages 1 or

2 (Duluth Area Fisheries file data).

probably will smolt within a short time. Fish that

fit these criteria should be stocked as soon as pos-

sible in the location to which imprinting is desired,

especially if returns to remote locations (away

from the hatchery outflow) are desired. Stockingsmaller fish at remote locations and larger fish at

the hatchery site may promote appropriate im-

printing. These recommendations may be less im-

portant in regions where the hatchery does not

discharge into the stocked watershed. Fish stocked

at remote locations should be placed some distance

upstream to enhance imprinting.

Imprinting to locations away from the hatchery

may be compromised in larger fish that have al-

ready partially imprinted and may have regressed

to a presmolt condition, especially if the season of

normal smolting is past. Growth of migratory rain-

bow trout should be controlled in the hatchery to

avoid production of very large (260 mm FL)

individuals. Upstream stocking of very large mi-

gratory rainbow trout should be avoided because

these fish may tend to residualize.

Chinook salmon smoltification appears to be in-

fluenced to some extent by temperature. Increasing

temperature early in the rearing process could help

to bring 100% of the age-0 fish to the minimum

71 mm FL by May. As the season of smoltification

approaches, chinook salmon that will be stockedin remote locations could then be maintained at 5–

7C to reduce imprinting in the hatchery and

stocked into water about 10–12C to enhance

smoltification and imprinting at that time. Those

destined for stocking at a hatchery location could

be held at 10–12C to enhance smoltification and

imprinting before stocking. Stocking at several lo-

cations or on several days from late May throughearly June may reduce immediate mass emigra-

tions of nonsmolts and smolts.

The ATPase activity assays were a useful tool

for defining the approximate size and time of

smolting in chinook salmon and migratory rain-

bow trout in this study. This knowledge will assist

with the effective use of hatchery stocks, enable

managers to make informed decisions regarding

hatchery supplementation in the presence of nat-

uralized stocks, and provide some explanations re-

garding the fate of juveniles that emigrate from

streams before smolting. This type of informationis needed not only to assist in making biologically

sound management decisions, but also to provide

supporting evidence that helps to explain these de-

cisions to the concerned public.

Acknowledgments

I thank Fred Tureson, manager of the French

River State Fish Hatchery, for suggesting this pro-

ject. Randall Hicks was extremely accommodating

in arranging for laboratory space and equipment

at the University of Minnesota in Duluth and,along with Arun Goyal, provided helpful advice

in developing the laboratory protocols. Thanks are

extended to Robin Schrock for advice and refer-

ence samples that were invaluable for setting up

the ATPase assay procedures. Donald Schliep and

Kenneth Olsen (Duluth Area Fisheries) were very

helpful in providing stocking information, age

data, and stream temperature data. Fred Tureson

and Mark Gottwald at the French River State Fish

Hatchery provided the hatchery fish for samples,

Kenneth Olsen provided fish from the French and

Knife river smolt traps, and Tracy Close (Duluth

Fisheries Research) assisted with sample collec-

tion. Charles Anderson, Darryl Bathel, Tracy

Close, Donald Schreiner, and Paul Wingate re-

viewed earlier drafts of this manuscript. This pro-

ject was funded in part by the Federal Aid in Sport

Fish Restoration (Dingell–Johnson) Program.

Completion Report, Study 654, D-J Project F-26-

R Minnesota. Reference to trade names does not

imply endorsement by the Minnesota Department

of Natural Resources.

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